Aspergillosis is a commonly reported fungal disease that results in illness and death in avian species.1–6 Species reportedly at increased risk of infection include raptors, waterfowl, poultry, and psittacine birds, specifically Amazon parrots and African grey parrots (Psittacus erithacus).1,4 Successful treatment of aspergillosis often requires long-term, systemic treatment with antifungals delivered IV, orally, topically, or via nebulization.3,4 Drugs commonly used include itraconazole, clotrimazole, fluconazole, voriconazole, and amphotericin B, but adverse effects seen with these drugs, including liver and kidney damage, can sometimes preclude their use in some species and animals. In addition, treatment failures have been reported with the use of itraconazole and voriconazole for infections caused by certain strains of Aspergillus fumigatus, the most commonly isolated Aspergillus organism.7
Terbinafine hydrochloride, an allylamine antifungal commercially available in topical and oral formulations, provides primarily fungicidal action with a broad range of in vitro activity.8 Terbinafine prevents fungal biosynthesis of ergosterol, necessary for cell membrane synthesis, via the inhibition of squalene expoxidase.5,6,9 Historically, terbinafine has been used for the treatment of dermatophytosis, but it has been found that terbinafine is useful in the treatment of refractory and systemic fungal infections, particularly aspergillosis.10,11 An increase in Cmax has been detected in humans when terbinafine is administered with food.12 Oral administration of terbinafine is generally tolerated better than is other available antifungal drugs, but adverse effects, including mild to moderate gastrointestinal tract discomfort, rash or urticaria, and malaise, can develop.10
Currently, terbinafine is not licensed for use in veterinary medicine but is routinely used in an extralabel manner to treat fungal infections, especially dermatophytosis in small animal medicine.13 Pharmacokinetics and dose recommendations for terbinafine have been reported for cats,13 dogs,14,15 horses,15 red-tailed hawks (Buteo jamaicensis),5 and penguins (Spheniscus demersus).6 The recommended dose of terbinafine is 22 mg/kg/d and 15 mg/kg/d for red-tailed hawks5 and penguins,6 respectively. We are not aware of any published doses derived from pharmacokinetic or pharmacodynamic studies for psittacine species such as Amazon parrots. Oral administration of doses of 15 mg/kg reportedly have been used clinically for the treatment of aspergillosis in psittacine birds with favorable results,16 but preliminary pharmacokinetic trials with that dose failed to yield measurable plasma concentrations in African grey parrots.17
Comparative in vitro testing has revealed that terbinafine is more effective against Aspergillus spp than are other drugs (including azole antifungal drugs and amphotericin B) commonly used to treat diseases attributable to those organisms.4,16,18 The elimination of terbinafine in humans requires < 5% of the total cytochrome P450 capacity of the liver, which may minimize adverse effects when terbinafine is used alone or in combination with other antifungal drugs. Terbinafine also has a better safety profile than the azole antifungals and amphotericin B, with few adverse effects reported in human and veterinary medicine.5,6,8,9
The purpose of the study reported here was to determine the pharmacokinetics of terbinafine after oral administration to Hispaniolan Amazon parrots (Amazona ventralis). These parrots were selected for use in the study because of their popularity as companion animals and the high frequency of aspergillosis in Amazon parrots, compared with the frequency of the disease in other species of psittacine birds.4 The size of the parrots also enabled us to safely collect an adequate volume of blood from each bird at all time points.
Materials and Methods
Animals—The study involved 6 apparently healthy adult Hispaniolan Amazon parrots; each parrot weighed between 274 and 329 g. All birds had a healthy appearance and unremarkable results for a physical examination, CBC, and plasma biochemical analysis during the 6 months preceding the study. No drugs had been administered to the birds during the 12 months preceding the study.
All birds were housed separately or in pairs in wire cages. The room was maintained at a constant temperature of 22°C with a photoperiod of 12 hours of light and 12 hours of darkness. The birds were fed a commercially available pelleted dieta and were provided water ad libitum; food was changed in the morning and offered continuously. The diet was periodically supplemented with fresh fruit and vegetables. The experimental protocol was approved by the University of Tennessee Institutional Animal Care and Use Committee.
Experimental design—Birds were orally administered a single dose of terbinafine (60 mg/kg) into the crop with a stainless steel feeding tube (time 0). To ensure a fed condition, approximately 6 mL of a commercially available gavage feeding formulab was placed directly into the crop immediately after drug administration. The feeding formula was administered via a 12-mL syringe attached to the same stainless steel feeding tube used to administer the drug (the tube was not removed between drug and food administration). Fooda was also offered continuously during the period following administration when blood samples were collected.
The terbinafine hydrochloride suspension (25 mg/mL) was created as previously described19 from terbinafine hydrochloride tablets (250 mg/tablet). Briefly, the tablets were ground with a mortar and pestle, and the powder was mixed with 2 suspension vehicles.c,d The suspension was mixed until uniform, labeled, and then stored in the refrigerator prior to administration. The terbinafine suspension was administered within 24 to 48 hours after it was created, which is well within the time that the suspension would remain stable.19
Venous blood samples were collected from each bird at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after drug administration. At each time point, each bird was manually restrained and a blood sample (0.25 to 0.3 mL) was collected from the right or left jugular vein by use of a 26-gauge needle attached to a 1-mL plastic insulin syringe. Each blood sample was immediately transferred to a lithium heparin blood tube and placed in a refrigerator for a maximum of 2 hours, after which samples were centrifuged (16,000 × g for 10 minutes) and the plasma decanted. Plasma was stored at −80°C until subsequent analysis. All samples were analyzed within 3 months after collection.
Measurement of plasma concentrations of terbinafine—Plasma samples were analyzed by personnel at the Clinical Pharmacology Laboratory at the University of Tennessee via a reverse-phase high-performance liquid chromatography method. The system consisted of a separation module,e absorbance detector,f and computer equipped with chromatography software.g Terbinafine was extracted from plasma samples via liquid extraction. Briefly, frozen plasma samples were thawed and mixed in a vortexer, and 100 μL of plasma was transferred to a clean screw-top test tube. Then, 75 μL of an internal standard (butenafine [1.0 μg/mL]) was added to each tube. Hexane (3 mL) also was added to each tube, and the tubes were mixed in a rocker for 20 minutes and then centrifuged for 20 minutes at 1,000 × g. The organic layer was transferred to a clean tube and evaporated to dryness with nitrogen gas. Samples were reconstituted in 250 μL of mobile phase, which was a mixture of 20mM phosphoric acid with 0.1% triethylamine (pH, 3.0) and acetonitrile (65:35), and 100 μL of each reconstituted sample was analyzed.
Compounds were separated on a C18 columng (4.6 × 100 mm, 5 μm) with a C18 guard columnh; the column was maintained at ambient temperature (approx 22°C). A mixture of 20mM phosphoric acid with 0.1% triethylamine (pH, 3.0) and acetonitrile (65:35) was used as the eluent. Flow rate was 1.1 mL/min. Absorbance was measured at 224 nm.
Standard curves for analysis of plasma concentration were created with calibration samples made by fortifying untreated, pooled Hispaniolan Amazon parrot plasma with terbinafine to achieve concentrations (range, 5 to 1,500 ng/mL) that yielded a linear result. Calibration samples were prepared in exactly the same manner as plasma samples obtained from the parrots after drug administration. Mean recovery for terbinafine was 95%, and intra-assay and inter-assay variability ranged from 4.5% to 8.5% and 0.93% to 8.5%, respectively. The lower limit of quantification was 5 ng/mL.
Pharmacokinetic analyses—Pharmacokinetics for terbinafine were calculated with software.i Mean plasma concentrations of terbinafine over time were plotted with software.i Values for plasma half-life, Cmax, Tmax, and AUC0-∞ were calculated from noncompartmental analysis. The area under the curve was calculated on the basis of the log-linear trapezoidal rule. Mean residence time was calculated as the total area under the first moment curve from time 0 to infinity/AUC0-∞.
Results
No adverse effects were detected during the study. Data for 1 bird were excluded because the results suggested a possible error in the dose of drug administered. Pharmacokinetic data for the remaining 5 birds were summarized (Table 1). Mean plasma concentrations of terbinafine over time were plotted (Figure 1). The Cmax ranged from 109 to 671 ng/mL, plasma half-life ranged from 6 to 13.5 hours, and Tmax ranged from 2 to 8 hours.
Mean ± SD plasma concentrations after oral administration of a single dose of terbinafine (60 mg/kg) to 5 Hispaniolan Amazon parrots (Amazona ventralis). Time of drug administration was designated as time 0. Birds were administered terbinafine, which was followed immediately by administration of a commercially available gavage feeding formula to ensure a fed state. Data for 1 other bird were excluded from analysis because of a possible error in the dose of drug administered.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.835
Mean ± SD plasma concentrations after oral administration of a single dose of terbinafine (60 mg/kg) to 5 Hispaniolan Amazon parrots (Amazona ventralis). Time of drug administration was designated as time 0. Birds were administered terbinafine, which was followed immediately by administration of a commercially available gavage feeding formula to ensure a fed state. Data for 1 other bird were excluded from analysis because of a possible error in the dose of drug administered.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.835
Mean ± SD plasma concentrations after oral administration of a single dose of terbinafine (60 mg/kg) to 5 Hispaniolan Amazon parrots (Amazona ventralis). Time of drug administration was designated as time 0. Birds were administered terbinafine, which was followed immediately by administration of a commercially available gavage feeding formula to ensure a fed state. Data for 1 other bird were excluded from analysis because of a possible error in the dose of drug administered.
Citation: American Journal of Veterinary Research 74, 6; 10.2460/ajvr.74.6.835
Pharmacokinetic values after oral administration of terbinafine hydrochloride (60 mg/kg) to fed Hispaniolan Amazon parrots (Amazona ventralis).
| Variable | Mean | Range |
|---|---|---|
| Terminal half-life (h) | 8.71 | 8.56–13.51 |
| Elimination rate constant (1/h) | 0.09 | 0.05–0.12 |
| Cmax (ng/mL) | 353 | 109–671 |
| Tmax (h) | 6.4 | 2.0–8.0 |
| AUC0-∞ (h·ng/mL) | 3,345 | 1,902–4,436 |
| MRT0-∞ (h) | 13.03 | 8.56–21.92 |
Results represent data for 5 birds; data for 1 other bird were discarded because of a possible error in the dose of drug administered.
MRT0-∞ = Mean residence time from time 0 to infinity.
Discussion
The objective of the present study was to determine whether oral administration of terbinafine to Hispanolian Amazon parrots at a dose consistent with doses used in other species would provide potentially therapeutic plasma concentrations. The in vitro MICs for Aspergillus flavus and A fumigatus have been reported as 0.01 to 0.5 μg/mL and 0.02 to 5.0 μg/mL, respectively.8 Target peak therapeutic plasma concentrations used for determining appropriate drug administration in red-tailed hawks have been extrapolated from the MIC for A fumigatus and were 1 to 4 μg/mL,5 but to our knowledge, in vivo therapeutic concentrations have not been established for the treatment of aspergillosis in any species of bird. The peak concentrations achieved by administration at a dose of 60 mg/kg in the present study were within the range for the in vitro MIC, but the maximum concentrations of all parrots failed to reach the therapeutic range of 1 to 4 μg/mL suggested for red-tailed hawks.
In a preliminary study, 3 Hispaniolan Amazon parrots from which food had been withheld were orally administered terbinafine at a dose of 30 mg/kg; the dose was selected on the basis of the fact that potentially therapeutic plasma concentrations were achieved in red-tailed hawks administered that dose of terbinafine5; however, the plasma concentrations thought to be therapeutic in red-tailed hawks were not reached in those 3 Hispanolian Amazon parrots. A 60 mg/kg dose in fed birds was chosen for the present study in an attempt to increase plasma concentrations. Absorption characteristics of terbinafine were not affected when the drug was administered with food to red-tailed hawks,5 but in humans, consumption of food resulted in an increase in plasma concentrations.12 In addition, ill birds infected with Aspergillus organisms often fail to ingest a sufficient number of calories to meet metabolic needs and are routinely fed via gavage to provide nutritional support at the time of medication administration. For this reason, in the opinion of one of the investigators (MJS), terbinafine pharmacokinetics in fed birds are likely more clinically relevant than are pharmacokinetics for unfed birds.
For the 60 mg/kg dose, Tmax ranged from 2 to 8 hours, which is similar to the Tmax of terbinafine when administered to other avian species.5,6 Substantial variation among the parrots was evident for Cmax (range, 109 to 671 ng/mL) and terminal half-life (5.99 to 13.51 hours), but these values were similar to values reported in other avian species.5,6 Unfortunately, data for 1 bird were excluded because of possible problems with the dose of drug administered.
Regurgitation is a reported adverse effect after terbinafine administration in avian species, but it has been observed only after frequent administration or administration of high doses.5 No adverse effects were detected for the Amazon parrots in the present study. Additional studies that include use of the dose of 60 mg/kg as well as higher doses and repeated administration of doses need to be performed to more fully assess the safety of terbinafine in this avian species.
Analysis of results of the present study suggests that higher doses of terbinafine (≥ 60 mg/kg) may be needed to achieve potentially therapeutic plasma concentrations in Amazon parrots. This is in contrast to the suggested doses of terbinafine for red-tailed hawks (22 mg/kg)5 or penguins (15 mg/kg).6 Pharmacokinetic studies of terbinafine in avian species have relied on in vitro–based determinations of MICs, and determining the in vivo concentrations of terbinafine necessary to treat aspergillosis in avian species would be extremely beneficial in future pharmacological studies. No adverse effects were observed in this study; however, additional trials with a larger number of birds that have been fed and, possibly, from which food has been withheld and additional pharmacokinetic and pharmacodynamic studies at higher and varied doses are warranted to evaluate safety and effects of multiple doses.
ABBREVIATIONS
| AUC0-∞ | Area under the plasma concentration-time curve from time 0 to infinity |
| Cmax | Maximum plasma concentration |
| MIC | Minimum inhibitory concentration |
| Tmax | Time to maximum plasma concentration |
Lafeber's Premium Daily Diet, Lafeber Co, Cornell, Ill.
Exact hand feeding formula, Kaytee Products Inc, Chilton, Wis.
Ora-Plus oral suspending vehicle (50 mL), Paddock, Minneapolis, Minn.
Ora-Sweet syrup (100 mL), Paddock Laboratories Inc, Minneapolis, Minn.
2695 separations module, Waters Corp, Milford, Mass.
2487 absorbance detector, Waters Corp, Milford, Mass.
Empower software, Waters Corp, Milford, Mass.
Symmetry Shield, Waters Corp, Milford, Mass.
WinNonlin, version 5.2, Pharsight Corp, Mountain View, Calif.
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